Enrichment of a Gonadotrope Population for Cell Specific Study

 描述（由申请方提供）：雌二醇-17 β（E2）是通过作用于下丘脑和垂体作用部位启动LH排卵前峰的关键内分泌信号。促性腺激素释放激素（GnRH）是下丘脑神经肽，它与位于垂体促性腺激素细胞上的GnRH受体结合，最终促使垂体分泌LH。因此，促性腺激素必须整合下丘脑和卵巢的信号以产生LH峰。尽管E2在调节垂体LH分泌和增强垂体对GnRH的敏感性方面起着关键作用，但我们对E2在垂体前叶水平发挥作用的机制的理解仍然有限。例如，虽然E2刺激GnRH受体（GnRHR）基因的表达是明确的，但促性腺激素内的潜在机制仍不明确，可能涉及核和膜信号传导事件，以及遗传和非遗传反应。不幸的是，由于促性腺激素占总垂体内分泌细胞群的约10%，由于几乎无法克服的信噪比，整个垂体对促性腺激素特异性分子事件的询问变得有问题。本提案的目标是开发一种基于腺病毒介导的基因转移和荧光激活细胞分选（FACS）的稳健方法，以从绵羊垂体中分离出富集的促性腺激素群体，从而对促性腺激素功能的关键介质（包括E2和GnRH）的基因和蛋白质表达进行定向和无偏倚分析。我们选择绵羊模型既是理论上的，也是技术上的。关于前者，我们认为，一个更完整的，相关的，和知情的分子平台，了解下丘脑和卵巢调节促性腺激素功能可以定义使用绵羊-不排除小鼠模型，但作为一个重要的补充。关于后者，可以从绵羊垂体中分离的促性腺激素的绝对数量将允许在单个动物中基因组和非基因组应答的稳健覆盖和复制。因此，我们建议融合腺病毒介导的基因转移和荧光蛋白的靶向表达，以产生一个可靠的方法，用于分离富集的羊促性腺细胞群体。如果成功，这项工作将为未来的研究建立技术基础，充分整合平行和互补的方法，以确定下丘脑，卵巢和促性腺激素代谢输入诱导的遗传和非遗传事件。鉴于E2或E2类似物的广泛治疗应用，对E2在整个身体中作用的分子事件的透彻理解与人类生育能力和人类健康和疾病高度相关。

 DESCRIPTION (provided by applicant): Estradiol-17β (E2) is the key endocrine signal that initiates the pre-ovulatory surge of LH by acting at both hypothalamic and pituitary sites of action. The secretion of LH from the pituitary is ultimately cued by the hypothalamic neuropeptide, gonadotropin-releasing hormone (GnRH), which binds to GnRH receptors located on gonadotrope cells in the pituitary gland. Thus, gonadotropes must integrate both hypothalamic and ovarian signals to mount the LH surge. Despite the key role of E2 in regulating pituitary LH secretion and enhancing pituitary sensitivity to GnRH, our understanding of the mechanisms by which E2 exerts its actions at the level of the anterior pituitary gland remains limited. For example, while it is unequivocal that E2 stimulates expression of the GnRH receptor (GnRHR) gene, the underlying mechanisms within the gonadotrope remain undefined and may involve both nuclear and membrane signaling events, as well as genotropic and non- genotropic responses. Unfortunately, since gonadotropes comprise approximately 10% of the total pituitary endocrine cell population, whole pituitary interrogation of gonadotrope specific molecular events becomes problematic due to an almost insurmountable signal to noise ratio. The goal of this proposal is to develop a robust methodology based on adenoviral mediated gene transfer and fluorescence activated cell sorting (FACS) to isolate an enriched population of gonadotropes from the ovine pituitary that will allow for both directed and non-biased analytics of gene and protein expression in response to key mediators of gonadotrope function including E2 and GnRH. Our choice of the sheep model is both theoretical and technical. In regard to the former, we believe that a more complete, relevant, and informed molecular platform for understanding hypothalamic and ovarian regulation of gonadotrope function can be defined using sheep -- not to the exclusion of mouse models, but as an important complement. In regard to the latter, the sheer numbers of gonadotropes that could be isolated from a sheep pituitary will allow robust coverage and replication of both genomic and non-genomic responses in a single animal. Thus, we propose to fuse adenoviral mediated gene transfer and targeted expression of fluorescent proteins to yield a reliable approach for isolating an enriched population of ovine gondadotrope cells. If successful, this work will establish the technical foundation for future studies that fully integrate parallel and complementary approaches to identify the genotropic and non-genotropic events induced by hypothalamic, ovarian and metabolic inputs to gonadotropes. Given the widespread therapeutic application of E2 or E2 analogs, a thorough understanding of the molecular events underlying E2 actions throughout the body is highly relevant to human fertility and human health and disease.